Effect of Par-4 reduction on GRP78 localisation in evCTB.

<p>HIPEC 65 cells (A- to D-) or primary evCTB (E-, F-) were transfected with control (ctrl) or Par-4 siRNA for 24 h. Cells were then trypsinized, plated and cultured for 48 h before protein extraction and Cell-ELISA, or incubated for 72 h for subcellular fractionation. A- Western blot of cytosolic and membrane proteins was performed under reducing conditions and probed with anti-GRP78, Par-4, Actin and GAPDH antibodies. B- Western blot of protein extracts (40 µg) was performed under reducing conditions and probed with anti-GRP78, Par-4 or GAPDH antibodies. C- Quantification of membrane GRP78 expression from 3 independent experiments of western blot. D- Cell-ELISA of GRP78 on HIPEC 65 cells. Data are expressed as the relative ratio of membrane (mb) over total GRP78 in percent. *p<0.05. E- Western blot of protein extracts (40 µg) from primary evCTB was performed under reducing conditions and probed with anti-GRP78, Par-4 or GAPDH antibodies. F- Cell-ELISA of GRP78 on primary evCTB. Data are expressed as the relative ratio of membrane (mb) over total GRP78 in percent. *p<0.05.</p

Effect of Par-4 on invasive property of trophoblastic cells.

<p>HIPEC 65 cells were transfected with control or Par-4 siRNA (A-) or control (ctrl) or Par-4 expressing plasmid (B-) for 24 h. Cells were then trypsinized and seeded on collagen I precoated transwell membranes for 48 h. Cells that invaded collagen were quantified by colorimetric assay. Data were expressed as the percentage of transfected cells that invaded the collagen-coated membrane relative to the control cells. C- Invasive properties of primary evCTB transfected with control (ctrl) or Par-4 siRNA. *p<0.05.</p

Sequence of primers used for qPCR.

<p>Sequence of primers used for qPCR.</p

Expression of Par-4 in trophoblastic cells.

<p>Immunohistochemistry of first trimester trophoblast with control IgG (a) or Par-4 antibodies (b). Magnification: x40.</p

Effect of forskolin treatment on GRP78 expression in trophoblastic cells.

<p>Primary first trimester or term CTBs and BeWo cells were treated or not with 100 µM forskolin for 96 h or 48 h respectively. A- Total RNA was then extracted, reverse transcribe andGRP78 cDNA was quantified by qPCR and normalized to cyclophilin A. B- Proteins (40 ug) were analysed by western blot with anti-GRP78 and anti-GAPDH antibodies. C- Membrane GRP78 expression was quantified by cell-ELISA in BeWo cells. Results are presented as mean ± SEM. n = 3 * p<0.05.</p

Demographic and clinical characteristics of the study groups.

<p>Demographic and clinical characteristics of the study groups.</p

Effect of GRP78 down regulation on hCG secretion.

<p>BeWo cells were transfected with GRP78 or control siRNA and seeded on gelatin layer and then treated or not with 100 µM forskolin for 48 h. hCG level in culture supernatant was determined by ELISA and normalized to protein extract concentration. n = 3, * p<0.05.</p

Role of GRP78 on fusion properties of BeWo cells.

<p>A- Cells were untreated (CTRL) or treated with anti-syncytin (AbSyn) or anti-GRP78 (AbC20) antibodies, 20 µM VST or CB106 for 48 h in the absence or presence of 100 µM forskolin. B- Cells were transfected with GRP78 or control siRNA for 48 h in presence or not of 100 µM forskolin. Fusion index was calculated for 3 independent experiments. * p<0.05.</p

Downregulation of GRP78 in Bewo cells.

<p>A- Cells were untreated (CTRL) or treated with 20 µM VST or CB106 for 48 h. B- Cells were transfected with GRP78 or control siRNA for 48 h. A–B Upper panel: Immunoblots of GRP78 and GAPDH. Lower panel: The intensity of the GRP78 bands from three independent experiments was quantified and normalized to GAPDH.</p

Role of GRP78 on phosphatidylserine flip of BeWo cells.

<p>A- BeWo cells were seeded on gelatin layer and treated or not with anti-syncytin (AbSyn) or anti-GRP78 (AbC20) antibodies, 20 µM VST or CB106 in the absence or presence of 100 µM forskolin for 48 h. B- BeWo cells were transfected with GRP78 or control siRNA and seeded on gelatin layer. The following day, cells were treated or not with forskolin for 48 h. Phosphatidylserine flip was evaluated by a colorimetric assay (APOPercentage). n = 3, * p<0.05.</p